Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

• the present invention relates to methods of identifying candidate compounds for regulating skeletal muscle mass or function or regulating the activity or expression of a corticotropin releasing factor-2 receptor (CRF 2 R).
• the invention also relates to methods for the treatment of skeletal muscle atrophy or methods for inducing skeletal muscle hypertrophy using CRF 2 R as the target for intervention and to metliods of treating muscular dystrophies using CRF 2 R and corticotropin releasing factor-1 receptor (CRF]R) as targets.
• Corticotropin releasing factor or hormone, CRF or CRH
• CRF Corticotropin releasing factor
• CRF is a major modulator of the body's responses to stress. This 41-amino acid peptide presides over a panoply of neuronal, endocrine, and immune processes as the primary regulator of the hypothalamus-pituitary-adrenal hormonal axis (HP A axis).
• CRF 2 R knockout mice in which the CRF 2 R gene has been ablated demonstrate an impaired food intake reduction following stimulation with urocortin, lack of vasodilation, but a normal stress response
• CRF 2 R knockout mice in which the CRF 2 R gene has been ablated demonstrate an impaired food intake reduction following stimulation with urocortin, lack of vasodilation, but a normal stress response
• CRF 2 R demonstrated that CRF 2 R is responsible for the hypotensive/vasodilatory effects of CRFR agonists and for the reduction in food intake observed following treatment of mice with CRFR agonists
• Skeletal muscle atrophy can also result from chrome conditions such as cancer cachexia, chronic inflammation, AIDS cachexia, chronic obstructive pulmonary disease (COPD), congestive heart failure, genetic disorders, e g . muscular dystrophies, neurodegenerative diseases and sarcopenia (age associated muscle loss) In these chronic conditions, skeletal muscle atrophy can lead to premature loss of mobility, thereby adding to the disease-related morbidity
• Muscular dystrophies encompass a group of inherited, progressive muscle disorders, distinguished clinically by the selective distribution of skeletal muscle weakness
• the two most common forms of muscle dystrophy are Duchenne and Becker dystrophies, each resultmg from the inheritance of a mutation in the dystrophin gene, which is located at the Xp21 locus
• Other dystrophies include, but are not limited to, limb-girdle muscular dystrophy which results from mutation of multiple genetic loci mcluding the p94 calpain, adhahn, ⁇ -sarcoglycan, and ⁇ - sarcoglycan loci, fascioscapulohumeral (Landouzy-Deje ⁇ ne) muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy
• the symptoms of Duchenne muscular dystrophy which occurs almost exclusively in males, include a waddlmg gait, toe walking, lordosis, frequent falls and difficulty in standm
• the mvention further provides methods for identifying candidate compounds that increase CRF 2 R expression comprising contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a CRF 2 R gene regulatory element and detecting expression of the reporter gene Test compounds that increase expression of the reporter gene are identified as candidate compounds for increasing CRF 2 R expression.
• the present invention relates to a method of determining whether those candidate compounds which increase CRF 2 R expression can be used to regulate skeletal muscle mass or function in vivo by administering a candidate compound to a non-human animal and dctermimng whether the candidate compound regulates skeletal muscle mass or function m the treated animal
• the mvention further provides for pharmaceutical compositions comprising a safe and effective amount of a CRF 2 R agonist and a pharmaceutically-acceptable carrier
• a pharmaceutical composition comprises a chimeric or human antibody specific for a CRF 2 R
• the pharmaceutical composition comprises a CRF or CRF analog, preferably urocortin II
• FIG. 5 demonstrates the anti-atrophy and hypertrophy inducing effects of sauvagine and urocortin (administered continuously by osmotic minipump) on the tibialis anterior muscle in the mouse sciatic nerve denervation-induced atrophy model.
• FIGS. 6A and 6B demonstrate the anti-atrophy effects of urocortin (administered subcutaneously, 2X daily) on the disuse-induced atrophy of the tibialis anterior muscle (FIG 6A) and of the medial gastrocnemius muscle (FIG 6B).
• FIG. 7 demonstrates in the anti-atrophy effect of sauvagine (administered subcutaneously, 2X daily), in the adrenalectomized rat sciatic nerve denervation-induced atrophy model, on the denervation-induced atrophy of the tibialis anterior (FIG. 7A), extensor digitorum longus (EDL) (FIG. 7B), soleus (FIG. 7C), medial gastrocnemius (FIG. 7D), and plantaris muscles.
• sauvagine induced hypertrophy of the non-denervated EDL muscle FIG.
• FIGS. 9A and B demonstrate that in a mouse leg casting disuse atrophy model, sauvagine had an anti-atrophy effect on the EDL and soleus muscle as measured by mass (FIG 9 A) or muscle function (FIG 9B).
• Antibody in its various grammatical forms, means immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen.
• Purified antibody means an antibody which has been partially or completely separated from the proteins and naturally-occurring organic molecules with which it is naturally associated.
• the preparation is at least 60% antibody, more preferably at least 75% antibody, more preferably at least 90% antibody, and most preferably at least 99%, by dry weight, antibody.
• Binding affinity means the propensity for a ligand to interact with a receptor and is inversely related to the dissociation constant for a specific CRF ligand-CRFR interaction.
• the dissociation constant can be measured directly via standard saturation, competition, or kinetics binding techniques or indirectly via pharmacological techniques involving functional assays and endpoints.
• CRFR means CRF ,R or CRF 2 R.
• CRF iR includes, but is not limited to, those receptors for which the cDNA or genomic sequence encoding the receptor has been deposited in a sequence database. These sequences include Accession Nos.: X72304, El 1431, L23332, 192584, T37068, T28968, Q81952, L23333, NM_004382, AF180301, T28970, L25438, L24096, 192586, Q81954, AH006791, NM_007762, X72305, AF054582, Y14036, AF229359, AF229361, AB055434 and L41563.
• CRF 2 R means any isoform of CRF 2 R from any animal species CRF 2 R has also been referred to as HM-CRF, CRF-RB , (Kishimoto, T , et al , Proc Natl Acad Sci USA, 92 1108- 1112 (1995) and Perrin, M et al Proc Natl Acad Sci USA 92 2969-2973 (1995))
• CRF 2 R receptor includes, but is not limited to, those receptors for which the DNA sequence encoding the receptor has been deposited in a sequence database These sequences include Accession Nos U34587, E12752, NM_001883, T12247, T66508, AF011406, AF019381, U16253, T12244, T28972, U17858, NM_009953, Y14037 and AF229360
• the nucleotide and protem sequences of these receptors are available from GenBank or Derwent and for convenience, representative sequences are given in the sequence listing herein
• CRFRs from a species other than those listed above, particularly mammalian species would be useful in the present mvention
• One of skill in the art would further recognize that by using probes from the known CRFR species' sequences, cDNA or genomic sequences homologous to the known sequence could be obtained from the same or alternate species by known clomng methods
• Such CRFiR are also included in the definition of CRFiR and such CRF 2 R are also included in the definition of CRF 2 R
• CRFR fusion proteins Fusions of a CRFiR or CRF 2 R polypeptide, or a CRFiR or CRF R polypeptide fragment to a non-CRFR polypeptide are referred to as CRFR fusion proteins.
• the non-CRFR polypeptide may be a signal (or leader) polypeptide sequence which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to another site (e.g., the yeast ⁇ -factor leader).
• non-CRFR polypeptide may be added to facilitate purification or identification of the CRFR (e.g., poly-His, or Flag peptide).
• CRFiR fusion proteins are also included within the definition of CRFiR and CRF 2 R fusion proteins are also included within the definition of CRFjR.
• CRF 2 R signal transduction pathway means any signaling pathway (e.g., cAMP, MAP kinase) or combination of signaling pathways that are modulated by the binding of endogenous or exogenous ligands to CRF 2 R.
• “Functional CRFRs” refers to CRFRs, which bind CRF or a CRF analog in vivo or in vitro and are activated as a result of ligand binding.
• Fusion gene means two or more DNA coding sequences operably associated so as to encode one hybrid protein.
• a “fusion protein” is the protein product of a fusion gene.
• two DNA sequences are said to be "operably associated” if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of a promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
• a coding sequence and regulatory sequences are operably associated when they are covalently linked in such a way as to place the transcription of the coding sequence under the influence or control of the regulatory sequences.
• a promoter region is operably associated with a coding sequence when the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript is capable of being translated into the desired protein or polypeptide.
• Percent identity means the percentage of nucleotides or ammo acids that two sequences have in common, calculated as follows To calculate the percent identity for a specific sequence (the query), the relevant part of the query sequence is compared to a reference sequence using the BestFit comparison computer program, Wisconsm Package, Version 10 1, available from the Genetics Computer Group, Inc This program uses the algorithm of Smith and Waterman, Advances in Applied Mathematics, Issue 2 482-489 (1981) Percent identity is calculated with the following default parameters for the BestFit program the scoring matrix is blosum62 cmp, the gap creation penalty is 8 and the gap extension penalty is 2 When comparmg a sequence to the reference sequence, the relevant part of the query sequence is that which is derived from a CRFR sequence For example, where the query is a CRFR/purification tag fusion protem, only the CRFR polypeptide portion of the sequence is aligned to calculate the percent identity score
• Polypeptide means any cham of ammo acids, regardless of length or post-translational modification (e g , phosphorylation or glycosylation)
• prophylactic treatment means preventive treatment of a subject, not currently exhibiting signs of skeletal muscle atrophy, in order to completely or partially block the occurrence of skeletal muscle atrophy
• One of skill m the art would recognize that certain individuals are at risk for skeletal muscle atrophy as discussed in the background section herein
• one of skill in the art would recognize that if the biochemical changes leading to skeletal muscle atrophy are appropriately regulated, that the occurrence of atrophy would be prevented or reduced m at-risk individuals For example, muscular dystrophy patients beginning treatment with corticosteroids are at risk for developmg skeletal muscle atrophy indicating that prophylactic treatment of such patients would be appropriate
• Regulation of skeletal muscle mass or function includes regulation of skeletal muscle mass, skeletal muscle function or both
• Reporter gene means a coding sequence whose product can be detected, preferably quantitatively, wherein the reporter gene is operably associated with a heterologous promoter or enhancer element which is responsive to a signal which is to be measured.
• the promoter or enhancer element in this context is referred to herein as a "responsive element”.
• a "therapeutically effective amount" of a substance is an amount capable of producing a medically desirable result in a treated patient, e.g., decreases skeletal muscle atrophy, increases skeletal muscle mass or increases skeletal muscle function, with an acceptable benefit: risk ratio; in a human or non-human mammal.
• FIG 2 shows that sauvagme inhibits denervation-induced atrophy of the tibialis anterior muscle m a mouse sciatic nerve denervation atrophy model
• a - water (control) B - sauvagine (0 1 mg/kg/d)
• C - sauvagine (0 3 mg/kg/d)
• D - sauvagme (1 0 mg/kg/d)
• * - p ⁇ 0 05 compared to water
• male mice were dosed with either sauvagine or vehicle control (physiological salme) by continuous infusion usmg an Alzet osmotic minipump at 5 ⁇ l/hr until the end of the experimental period (without additional theophyllme)
• the daily delivered dose of sauvagine is mdicated above Mimpump implantation was performed at the time of sciatic nerve denervation On day mne the tibialis anterior muscle was removed and weighed to deten ne the degree of atrophy
• FIG. 7 demonstrates that sauvagine inhibits denervation-induced atrophy of the tibialis anterior (FIG. 7 A), EDL (FIG. 7B), soleus (FIG. 7C), medial gastrocnemius (FIG. 7D), and plantaris , muscles.
• sauvagine caused statistically significant hypertrophy of the non-denervated EDL muscle (FIG. 7B).
• CRFiR, CRF R, CRF and CRF analogs can be prepared for a variety of uses, including, but not limited to, the generation of antibodies, use as reagents in the screening assays of the present invention, and use as pharmaceutical reagents for the treatment of skeletal muscle atrophy. It will be clear to one of skill in the art that, for certain embodiments of the invention, purified polypeptides will be most useful, while for other embodiments cell lines expressing the polypeptides will be most useftil. For example, in situations where it is important to retain the structural and functional characteristics of the CRFR, e.g., in a screening method to identify candidate compounds which activate CRFRs, it is desirable to use cells which express functional CRFRs
• recombinant DNA technology m the preparation of CRFi , CRF 2 R, or of cell lines expressing these polypeptides is particularly contemplated. Such recombinant methods are well known in the art.
• an expression vector that comprises a nucleic acid which encodes the polypeptide of interest under the control of one or more regulatory elements is prepared.
• cells useful m this assay are the same as for the CRFR binding assay described above, except that cells utilized in the activation assays preferably express a functional receptor which gives a statistically significant response to CRF or one or more CRF analog
• cells can be engineered which express CRFRs contaimng the ligand binding domain of the receptor coupled to, or physically modified to contam, reporter elements or to interact with signaling proteins
• a wild-type CRFR or CRFR fragment can be fused to a G-protem resulting in activation of the fused G-protem upon agomst bmdmg to the CRFR portion of the fusion protem (Siefert, R et al , Trends Pharmacol Sci 20 383-389 (1999))
• the cells should also preferably possess a number of characteristics, depending on the readout, to maximize the inductive response by CRF or the CRF analog, for example, for detecting a strong induction of
• the leg casting disuse atrophy model involves castmg one hind leg of an animal from the knee down through the foot Typically, muscle mass is reduced 20-40% after ten days of casting
• test compounds are admmistered by mjection or by continuous infusion via implantation of an osmotic mimpump (e g , Alzet, Palo Alto, CA) to determine their effect on leg casting induced skeletal muscle atrophy
• an osmotic mimpump e g , Alzet, Palo Alto, CA
• the compounds for use according to the present invention may be delivered by, but not limited to, the following forms liquid, powder, gel or in the form of an aerosol spray utilizing either pressurized or non-pressunzed propellants in either premeasured or non-premeasured doses
• the pharmacologically active compound may be formulated with appropriate fillers, vehicles, preservatives, buffers, etc
• the phannacologically active compound may be formulated with acceptable physiological carriers, preservatives, etc and be prepared as suspensions, solutions, emulsion, powders ready for constitution, etc for either bolus injection or infusion Doses of these compounds may be administered by a variety of technologies mcludmg hypodermic needles, high pressure devices, etc
• the pharmacologically active compound may be formulated with acceptable physiological earners, preservatives, etc for delivery as suppositories, enemas, etc
• the pharmacologically active compound may be formulated with acceptable physiological earners, preservatives, etc for delivery as s
• the clones are characterized for both CRF binding and CRE-LUC activation following exposure to CRF as described in Example 2 and Example 3.
• Cells expressing the hCRF 2 R receptor at an appropriate level and which are appropriately coupled to the CRE-LUC reporter system are then utilized for further analysis.
• Receptor Activation Assay Receptor activation analysis is performed by seedmg the HEK293/CRE-LUC/pIRESneo/ hCRF 2 R cells of Example l to Packard View Plate-96 (Packard Inc , CA) Cells are seeded m DMEM medium containing 10% fetal bovine serum, penicillin/streptomycin solution, L-glutamme, and non- essential ammo acid at 37°C in a 5% carbon dioxide/95% air atmosphere and incubated overnight The medium is then removed and replaced with DMEM (Life Technologies, Rockville, MD) contaimng 0 01% bovme albumm fraction V (SIGMA, St Louis, MO) contaimng the compound of interest The cells are then incubated for four hours at 37°C in a 5% carbon dioxide/95% air atmosphere after which the medium is removed and the cells are washed twice with Hanks Balanced Salt Solution (Life Technologies, Rockville, MD) Lysis Reagent (Promega).
• This assay is perfonned by seeding the HEK293/ CRE-LUC/pIRESneo/hCRF 2 R receptor cells mto Packard View Plate-96 (Packard Inc , CA) Cells are seeded in DMEM medium contaimng 10% fetal bovine serum, penicillin/streptomycin solution, L-glutamme, non-essential ammo acid, and saturating amounts of CRF at 37°C m a 5% carbon dioxide/95% air atmosphere and incubated for 48 hours The medium is then removed and replaced with DMEM (Life Technologies, Rockville, MD) contaimng 0 01% bovme albumin fraction V (SIGMA, St Louis, MO) and CRF m addition to the compound of interest The cells are then incubated for four hours
• Example 6 Screens to identify candidate compounds that increase hCRF 2 R expression
• oligonucleotides one contaimng the 5 ' end of the promoter region (5 ' oligonucleotide) and one containing the 3 ' end of the promoter region mcluding the transcriptional start site (3' oligonucleotide) are synthesized These oligonucleotides also contain restriction endonuclease sites which are not present in the hCRF 2 R gene regulatory region with one umque site in the 5' oligonucleotide and a different unique restriction endonuclease site in the 3' oligonucleotide The 5' and 3' oligonucleotides are used for PCR amplification of the hCRF 2 R gene regulatory region from human DNA (
• Example 7 Screens to identify compounds that mcrease human CRF expression
• the methods for identifying compounds that increase human CRF (hCRF) expression are essentially identical to those for identifying compounds which mcrease hVPAC 2 receptor expression except the regulatory region used is that for the hCRF gene
• the sequence containing the regulatory region of the hCRF gene, beginning far enough upstream of the transcriptional initiation site to contain all the regulatory elements necessary for physiological expression of the hCRF gene in the appropriate tissue is retrieved from the human genome database
• Two oligonucleotides, one containing the 5 ' end of the regulatory region (5 ' oligonucleotide) and one containing the 3 ' end of the regulatory region mcluding the transcriptional start site (3' oligonucleotide) are synthesized These oligonucleotides also contain restriction endonuclease sites which are not present in the hCRF gene regulatory region with one umque site m the 5 ' oligonucleotide and a different unique restriction endonuclease site in the 3
• Fully human monoclonal antibodies which activate the hCRF 2 R are produced by first generating recombinant hCRF 2 R protein as follows The procedure from Example 1 is followed to obtain the hCRF 2 R PCR product. This hCRF 2 R PCR product is then cloned into the pHAT20 vector (Clonetech Inc., Palo Alto, CA, USA) by first cutting the hCRF 2 R gene PCR product and the pHAT20 vector with the appropriate restriction endonucleases so that the 5' and 3' restriction endonuclease sites are ready for ligation.
• Ligation of the pHAT20 vector DNA to the hCRF 2 R gene PCR product DNA is accomplished using DNA ligase from the AdvantAgeTMPCR Cloning Kit (Clonetech Inc., Palo Alto, CA, USA) according to the manufacturer's recommendations.
• the ligated vector/insert construct is then used to transform TOPI OF' competent E. coli cells (Clonetech Inc., Palo Alto, CA, USA). Transformed cells are plated on LB plus ampicillm containing agar and ampicillin resistant colonies are selected for further analysis.
• hCRF 2 R -pHAT20 vector DNA is then used for additional PCR cloning by utilizing a 5' oligonucleotide containing the beginning of the HAT sequence and a unique restriction endonuclease site not present in the hCRF 2 R -pHAT20 construct and the 3' hCRF 2 R oligonucleotide utilized previously.
• the oligonucleotide primers are used to PCR amplify the HAT- hCRF 2 R fusion gene from the hCRF 2 R -pHAT20 construct and the PCR product is purified as described above.
• the HAT- hCRF 2 R fusion gene PCR product is then utilized for cloning into the pBacPAK8 vector using the BacPAK Baculovirus Expression System from Clonetech (Clonetech Inc., Palo Alto, CA, USA).
• the ligation of the HAT- hCRF 2 R fusion gene into the pBacPAK8 vector is essentially as described above.
• the virus is then amplified in Sf21 cells, the harvested virus titered, and the titered virus used for large scale infection of Sf21 cells utilizing BacPAK Insect Cell Media - all according to the manufacturers recommendations (Clonetech Inc., Palo Alto, CA, USA).
• Recombinant HAT-CRF 2 R fusion protein is then purified using the TALON® CellThru Purification Kit from Clonetech (Clonetech Inc., Palo Alto, CA, USA) usmg conditions recommended by the manufacturer. Briefly, infected Sf21 cells are harvested 48 hours after infection and sonicated in extraction/loading buffer. The cell lysate is then put through a TALON® CellThru column.
• the column is washed twice with extraction/loading buffer and the bound HAT- hCRF 2 R protein is eluted with elution buffer.
• the eluted protein is analyzed by SDS-PAGE for integrity and protein concentration is quantitated using the Bio-Rad SDS-PAGE system and protein quantitation systems according to the manufacturer's recommendations (Bio-Rad Laboratories, Hercules, Ca).
• Purified HAT- hCRF 2 R fusion protein is then used for immunizing XenoMouse animals (Abgenix Inc., Fremont, CA) for human monoclonal antibody production as follows.
• Serum is obtained from vaccinated annuals and utilized in an antigen capture ELISA utilizing purified HAT- hCRF 2 R fusion protein to detect antibodies to the HAT- hCRF 2 R protein by coating polystyrene ELISA plates (Corning Glass Works, Corning, NY) with HAT- hCRF 2 R fusion protein, blocked with PBS-1% BSA, washed and incubated at 37°C for 1 hour with a 1:50 dilution of the serum samples. After washing 5 times with PBS, the plates are incubated at 37°C for 1 hour with alkaline phosphatase-conjugated goat antibodies to human immunoglobulin G.
• Hybridomas are generated by fusion of splenic cells from the XenoMouse animals with nonsecreting myeloma cell line NSA-bcl 2 using a 4: 1 ratio of spleen cells to NSA-bcl2 cells in the presence of 30% polyethylene glycol PEG1450.
• Fused cells are individually cloned by limiting dilution into 96 well plates and cultured in RPMI-1640 medium containing 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, L-glutamine, 100 u/ml penicillin-streptomycin and hypoxanthine-aminopterin-thymidme (all from Life Teclinologies, Rockville, MD).
• supernatants from the hypoxanthine-aminopterin-thymidine selected hybridomas were screened for human antibody production by ELISA as described previously.
• Hybridomas which produce human antibodies to the HAT- hCRF 2 R fusion protein are selected for large scale antibody production.
• Monoclonal antibodies are purified by Protein G-Sepharose chromatography. Briefly, the supernatant from cultured hybridoma clones is loaded onto a Protein G-Sepharose column (SIGMA, St. Louis, MO) in loading buffer, washed 3 times and the IgG is eluted with elution buffer. These antibodies are then used for screening to evaluate hCRF 2 R activation (agonism) potential. This is accomplished using the methodology as outlined in Example 3. Those human monoclonal antibodies which demonstrate agomst activity toward the hCRF 2 R are designated candidate compounds
• the extensor digitorum longus (EDL) and soleus muscles are removed, tendon-to-tendon from the casted mouse leg A silk suture is tied to each tendon of the isolated muscles and the muscles are placed mto a plexiglass chamber filled with Ringer solution (137 mM sodium chloride, 24 mM sodium bicarbonate, 11 mM glucose, 5 mM potassium chloride, 1 mM magnesium sulfate, 1 mM sodium phosphate, 0 025 mM tubocurarme, all at pH 7 4 and oxygenated with 95% oxygen/5% carbon dioxide) constantly bubbled with 95 % oxygen 5% carbon dioxide mamtained at 25° C Muscles are aligned horizontally between a servomotor lever arm (Model 305B-LR Cambridge Technology Inc , Watertown MA, USA) and the stainless steel hook of a force transducer (Model BG-50, Kuhte Semiconductor Products Inc , Leonia, NJ, USA) and field stimulated by pulses transmitted between two platinum electrode
• Example 10 Therapeutic treatment of skeletal muscle atrophy usmg a human antibody that is an agonist of the hCRF 2 R receptor
• a human male subject weighmg 50 kg and having sigmficant muscular atrophy of the arms and legs due to prolonged bed rest, is treated to reverse the skeletal muscle atrophy
• 15 mis of an aqueous solution of pH 6 comprismg an activating antibody of the CRF 2 R receptor is admimstered to the subject via intravenous injection
• the solution comprises the following
• the subject At the end of the treatment period, the subject exhibits measurable increases of muscle mass, strength and mobility of the arms and legs
• Example 11 Prophylactic treatment of skeletal muscle atrophy using a human antibody that is an agomst of the hCRF 2 R receptor
• the subject At the end of the treatment period, the subject exhibits measurable preservation of muscle mass, strength and mobility of the arms and legs as compared to the subject's expected status without antibody therapy
• Example 12 Prophylactic treatment of skeletal muscle atrophy using a human antibody that is an agonist of the CRF2R receptor
• a human female subject weighing 45 kg undergoes a casting procedure to treat a simple fracture of the humerus after a fall.
• the subject is treated to prevent atrophy of the skeletal muscle of the affected ami and shoulder due to disuse and limited use during fracture healing.
• 13 ml of pH 6.0 comprising the anti-hCRF 2 R receptor is administered to the subject via intravenous mjection.
• the solution comprises the following:
• the subject At the end of the treatment period, the subject exhibits measurable preservation of muscle mass, strength and mobility of the affected arm and shoulder and a reduced course of physical therapy as compared to the subject's expected status and follow-up treatment without antibody therapy.
• Example 13 Prophylactic treatment of skeletal muscle atrophy using urocortin-II
• a human female subject weighing 60 kg is admitted to the hospital in a comatose state.
• the subject is treated by this method to prevent atrophy of the skeletal muscle of the entire body due to disuse in the comatose state.
• the subject is administered, via slow intravenous infusion, approximately 500 ml of an aqueous solution that is prepared by addition of 5 ml of the following stock solution to 500 ml of sterile saline:
• the subject exhibits measurable preservation of skeletal muscle mass and ftinction, and reduced physical therapy needs during the coma and after regaining consciousness, as compared to the subject's status without drag therapy
• Example 14 Therapeutic treatment of a patient with Duchenne Muscular dystrophy using CRF
• a male subject weighing 40 kg with an existing diagnosis of Duchenne's Muscular Dystrophy is treated with a compound that exhibits CRFl-R and CRF2-R agomsm over a similar dose range
• the subject is treated with a sustained-release, depot formulation of the compound in order to improve or retain muscle strength and function over the progression of the disease Specifically once each month the subject is administered, via intramuscular injection, 3 ml of an aqueous solution of pH 6 0 comprising the following
• the subject experiences either an improvement or an attenuation of the decline of muscle strength or muscle function m timed-function evaluations as compared to that exhibited durmg the natural progression of the disease

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